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CD3/CD28/DLL4 magnetic bead and preparation method and application thereof

A technology of magnetic beads and antibodies, applied in the biological field, can solve the problems of easy drug resistance of tumors, toxic and side effects, etc., and achieve the effects of controllable product quality, obvious effect, and convenient use

Pending Publication Date: 2021-12-24
苏州东岭生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Traditional chemotherapeutic drugs are prone to drug resistance and serious side effects in the treatment of tumors

Method used

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  • CD3/CD28/DLL4 magnetic bead and preparation method and application thereof
  • CD3/CD28/DLL4 magnetic bead and preparation method and application thereof
  • CD3/CD28/DLL4 magnetic bead and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: A method for amplifying and producing human effector CD8 + Preparation method and application of CD3 / CD28 / DLL4 magnetic beads for T lymphocytes;

[0049] 1) Preparation of CD3 / CD28 / DLL4 functional magnetic beads: Take 1*10^6~5*10^6 magnetic beads, wash the magnetic beads twice with MEST buffer (1 mL each time), add the pre-configured EDC solution , Vortex and mix well, incubate at 25°C for 20-60 minutes, mix the activated magnetic beads with the following protein solution and incubate: 25 μg anti-human-CD3 antibody solution + 25 μg anti-human-CD28 antibody + 10 μg DLL4 recombinant protein solution at 25°C Continue Incubate for 2-4 hours, avoid the magnetic beads from settling during the incubation period, then block the magnetic beads with MEST solution containing BSA for 0.5-2 hours, the blocking temperature is 25°C, and finally wash the magnetic beads 3 times with TBST (1 mL each time), and then store them After washing the magnetic beads for 3 times, s...

Embodiment 2

[0052] Example 2: CD3 / CD28 / DLL4 functional magnetic bead activation efficiency test;

[0053] 1) Take 0.2M purified T lymphocytes, add T cell special medium to adjust the total volume of cell suspension to 200 μL;

[0054] 2) Add the CD3 / CD28 / DLL4 magnetic beads prepared in Example 1, and wash the magnetic beads once with 1mL F-PBS before use;

[0055] 3) Spread the mixture of cells and magnetic beads on a 96-well plate and place in CO at 37°C 2 Cultivate overnight in an incubator;

[0056] 4) The next day, observe the state of the cells and the formation of clones under a microscope, and the results of the photos are attached figure 2 shown. figure 2 It shows that activated by CD3 / CD28 / DLL4 magnetic beads, the magnetic beads bind to form cell-magnetic bead complexes around the cells, and T lymphocytes have obvious proliferating clone populations, while the morphology of cells in the wells without magnetic beads does not change , and there is no proliferating clonal popu...

Embodiment 3

[0058] Example 3: Verification of cell expansion after activation of T lymphocytes by CD3 / CD28 / DLL4 magnetic beads;

[0059] 1) The T lymphocytes collected in Example 2 and activated overnight by CD3 / CD28 / DLL4 magnetic beads were kept in 37°C CO 2 Cultivated in an incubator;

[0060] 2) Take out the cells on the third day of culture, count them, take 0.1M cells and resuspend them in 1.5mL T cell special medium, spread them on a new 24-well plate and continue to culture them in a CO2 incubator at 37°C;

[0061] 3) Take out the cells on the seventh day of culture, count them, take 0.1M cells and resuspend them in 1.5mL T cell special medium, spread them on a new 24-well plate and continue to store them in 37°C CO 2 Cultivated in an incubator;

[0062] 4) Take out the cells on the tenth day of culture, count them, and calculate the cell expansion multiples. The expansion results are as attached Figure 4 shown; Figure 4 showed that CD3 / CD28 / DLL4 magnetic beads activated cult...

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Abstract

The invention discloses a CD3 / CD28 / DLL4 magnetic bead. The surface of the magnetic bead is coupled with an anti-human CD3 antibody, an anti-human CD28 antibody and a recombinant human DLL4 protein. The invention also discloses a preparation method and application of the CD3 / CD28 / DLL4 magnetic bead. When the CD3 / CD28 / DLL4 magnetic bead amplifies T lymphocytes, TCR on the surfaces of the T lymphocytes can be combined with anti-CD3 antibody cross-linked on the surfaces of the magnetic bead, and a first signal for activating the T lymphocytes is provided; cD28 on the surface of the T lymphocytes can be combined with the anti-CD28 antibody cross-linked on the surface of the magnetic bead to provide a second signal for activating the T lymphocytes; and the DLL4 can effectively promote the differentiation of CD4 + T cells into Th1 effector cells and promote the differentiation of CD8 + T cells into cytotoxic CD8 + T effector cells. The proportion of CD8 + T cells in the T cell mixture generated by CD3 / CD28 / DLL4 magnetic bead culture exceeds that of the T cell mixture obtained by traditional CD3 / CD28 magnetic bead amplification, and the generated CD8 + T cells can secrete higher-level killing factors when encountering antigen stimulation.

Description

technical field [0001] The invention relates to a CD3 / CD28 / DLL4 magnetic bead, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Traditional chemotherapy drugs in the treatment of tumors have the problems of easy drug resistance and serious side effects. Immunotherapy, as an emerging cutting-edge science and technology, has successfully cured many cases of refractory, easy-to-relapse, and hopeless tumor patients around the world, thus becoming a new star in the field of tumor treatment. T cell immunotherapy (chimeric antigen receptor CAR-T, T cell receptor TCR-T, etc.) is an important system in immunotherapy. Compared with traditional chemotherapy drugs, its advantages are strong specificity, low side effects, long-lasting therapeutic effects. In 2017, the US Food and Drug Administration approved the first CAR-T for clinical treatment worldwide; in 2021, the China Food and Drug Administration also approve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783H01F1/42H01F41/00
CPCC12N5/0636H01F1/42H01F41/00C12N2501/515C12N2501/51C12N2501/998C12N2501/2302C12N2500/44
Inventor 孟丽君张珍琦邓猛猛胡绍燕
Owner 苏州东岭生物技术有限公司
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